Vitamin A is an essential nutrient required not only for vision but also for the proper differentiation of many cell types in the body. This proposal seeks to define and study the molecular apparatus of the intestinal absorptive cell that carries out the necessary metabolism and movement of vitamin A during its transit of that cell during absorption. Particular elements under study are the enterocyte-specific retinol-binding protein, cellular retinol-binding protein II [CRBP(II)], a brush-border retinyl ester hydrolase, a brush-border retinol transporter, microsomal retinal reductase, and microsomal lecithin-retinol acyltransferase (LRAT). The specific aims are to: 1.) Purify the brush-border retinyl ester hydrolase for physical and biochemical characterization and for production of antiserum for radioimmunoassay and immunolocalization. Its ontogeny and distribution along the gastrointestinal tract in rat and other species will be determined. Its physiological role will be tested by comparing its properties to the abilities of rat everted gut sacs and the human intestinal Caco-2 cell line to utilize retinyl esters. 2.) Characterize retinol transport in both rat everted gut sacs and the Caco-2 cell. Possible coupling of the putative retinol transporter with both the brush border retinyl ester hydrolase and with CRBP(II) will be tested. Evidence for specific uptake mechanisms for retinol bound to several retinol binding proteins will be sought. 3.) Purify and characterize microsomal retinal reductase and produce antiserum for immunolocalization. Its physiological function will be tested by comparing its developmental pattern and the stereospecificity of its reduction of retinal to the abilities/properties observed for everted gut sacs and Caco-2 cells. 4.) Purify and characterize LRAT and produce antiserum for radioimmunoassay and for immunolocalization, with particular reference to the localization of retinal reductase. Possible channeling of retinoid from the reductase to LRAT will be tested. The source of substrate lecithin will be examined by testing the potential role of phospholipid transfer proteins as substrate carrier.